Virulence-associated factors are molecules involved in the virulence or pathogenicity of bacteria. Such factors are often associated for example, with gastrointestinal infections. Bacterial toxins are common virulence-associated factors.
Shiga toxin is the most well known member of a family of cytotoxins called "Shiga-like" toxins which are known virulence-associated factors. Shiga toxin is a multimeric protein toxin which is a potent inhibitor of eukaryotic protein synthesis. The toxin has one 32,000-mw polypeptide (the A chain) and five copies of a 7,600-mw polypeptide (the B chain). It is believed that the A chain is responsible for the toxin's ability to inhibit eukaryotic protein synthesis and the B chain is the receptor-binding moiety. The B chain recognizes cell-surface glycolipids having a terminal .alpha.-D-Gal.rho.-(1-4)-D-Gal.rho. portion (Acheson et al., The Journal of Infectious Diseases 1990:161:134-137). These toxins have been found in a variety of bacteria including E. coli, Vibrio cholerae, Shigella dysenteriae and other species of Shigella.
Shiga-like toxins I and II (hereinafter SLT I and SLT II) have been well characterized. It has been reported that SLT I differs from Shiga toxin by one amino acid in the A chain. It further has been reported that the enzymatic activity and binding specificity of Shiga toxin and SLT I are identical (Acheson et al., cited supra; Stockbrine et al., Infect Immun. 1986:53:135-140). SLT II, however, has been shown to differ in primary sequence from Shiga toxin and has only 56% amino acid homology . SLT II and Shiga toxin also share the same enzymatic activity and binding affinity for cell-surface glycolipids (Acheson et al., cited supra; Jackson et al., FEMS Microbiol. Lett 1987; 44:109-114).
SLT-producing E. coli (most frequently E. coli serotype 0157:H7) has been implicated in the pathogenesis of diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). HUS may result in microangiopathic hemolytic anemia, thrombocytopenia, and/or acute renal failure. HUS is the most common cause of acute renal failure in children. It has a 5-10% mortality rate in children and causes persistent disability in one third of patients. The case definition of TTP differs from HUS through the inclusion of fever and neurologic complications. The SLT-producing E. coli recently have emerged as the human pathogen associated with outbreaks of bloody diarrhea as well as cases of HUS and TTP in North America and Europe. The most common type of HUS develops in children following a diarrhea prodrome which is often bloody. SLT-producing E. coli including 0157:H7 are the most common pathogens found in such cases.
The infecting organisms have been found primarily in cattle (particularly dairy cows), milk, and meat products. E. coli 0157 was first recognized in food products provided by fast-food hamburger chains. The organisms appear to be highly infectious as documented in studies of outbreaks occuring in day care, nursing homes, school, and other institutional settings.
The majority of SLT producing E. coli strains are indistinguishable from E. coli in the normal fecal flora. A routine stool culture, therefore, does not detect this group of pathogens. The most predominant strain, E. coli. 0157:H7, ferments sorbitol slowly or not at all and therefore can be detected on special sorbitol-containing MacConkey agar medium. The sorbitol-negative colonies can be tested serologically using antisera to 0157 lipopolysaccharide and H7 flagellar antigens. Following isolation and identification of this serotype, the strains can be tested with the 4-methylumbelliferyl-.beta.-D-glucuronide (MUG) test, another biochemical test, which has shown good correlation with toxin production. The strain is tested for production of SLT by enzyme-linked immunosorbent assay (ELISA), DNA probe or immunoblot technology to confirm pathogenicity. Presently, strains are sent to the Center of Disease Control for toxin confirmation testing.
If an individual is suspected of being infected by E. coli producing SLT I or SLT II, it is desirable to test stool specimens at the earliest stage possible because the chance of successful culture and isolation of this pathogen decreases with time. Although the 0157 serotype can be detected by biochemical and immunological testing in the laboratory, over 50 other serotypes, known to produce toxin, now go undetected. The specialized tests described above, ELISA, DNA probe and immunoblot are carried out only in a handful of research facilities. Tissue culture assays have been proposed, however, such testing requires antisera against the toxins to demonstrate specific neutralization, and up to four days to detect the specific toxin reaction on the cells. These assays are expensive, tedious and labor intensive. The tissue culture assays require specialized facilities, equipment and reagents and, therefore, are not desirable for routine testing purposes.
SLT-neutralizing antibodies also are demonstrable during infection by SLT producing E. coli. A fourfold increase in titer has been demonstrated but a serologic test directed towards the antibodies has, to date, demonstrated only low sensitivity. Serologic tests directed towards these antibodies may be appropriate for epidemiologic investigations of outbreaks or to verify recent infection in HUS or TTP patients if the infecting organism or toxin were not found in the stool, but presently available serologic testing is not sensitive enough to be an efficient routine diagnostic test.
The detection of bacterial toxins such as Shiga-like toxins is challenging because the toxins have to be released from the bacteria prior to being detected. Lysing or releasing agents capable of releasing the toxins from the bacteria may adversely effect the structure of the toxins such that the toxins can not be recognized. There exists a need for better and more accurate assays for Shiga-like toxins. A simple and sensitive assay useful, for example, on stool samples could eliminate the need for tissue culture assays or serological testing and would satisfy a longfelt clinical need.